PROJECT SUMMARY/ABSTRACT: Objectives and Specific Aims: Inhalation of nitric oxide (NO) has been used as a treatment for pulmonary hypertension in children and adults. Wider use of this therapy is significantly limited, however, because the current delivery system is cumbersome (about 45 kg/cylinder) and expensive (about $180/hour). The objective of the proposed project is to test the purity and safety of NO generated by pulsed electrical discharges from air in our prototype, serving as a simple and economical source of NO for inhalation therapy in the hospital or outpatient setting. Based on our preliminary studies, two major aims for this project are proposed. In Aim 1, we will purify plasma generated NO and create a reliable backup NO generator. This includes identifying and quantifying metal particles during NO generation, evaluating the capacity of a single HEPA filter to block metal particles, and developing and building a complete backup system for safe delivery of NO. In Aim 2, we will study the pulmonary and systemic effects in awake mice breathing electrically generated NO for 30 days, and in awake lambs for 7 days. Research Design and Methods: We will test the purity and safety of NO generated by our current prototype in vitro and in vivo, to assure continuous and reliable delivery of NO. A couple of state of the art equipment will be employed for the proposed studies. The in vitro study includes using scanning electron microscopy (SEM), energy dispersive X-ray (EDX), and inductively coupled plasma mass spectrometry (ICP-MS) to identify and quantify metal particles, chemical compositions produced during NO generation. In in vivo study, we will employ ICP-MS to measure any metal particles in the lungs of animals exposed to electrically generated NO for prolonged periods. We will study any adverse pulmonary and systemic effects in mice and lambs after breathing electrically generated NO for 30 days and 7 days, respectively. We will employ immunohistochemical staining, biochemical, and molecular biological approaches to evaluate tissue injury, organ function, inflammatory response, and oxidative stress in mice and lambs after exposed to electrically generated NO. A chemiluminescence analyzer (Sievers Nitric Oxide 280i, GE Power and Water, Boulder, CO) will be used to measure the level of NO. An NO2 to NO converter (AMP Cherokee, Fuquay Varina, NC) connected with Sievers 280i will be used to detect NO2 levels above 2 ppm. To obtain accurate measurements of NO2 at levels below 2 ppm, a visible light (450 nm) absorption with cavity attenuated phase shift technology (Aerodyne Research Inc., Billerica, MA) will be employed. An EC 9810 series UV Ozone Analyzer (American Ecotech, Warren, RI) will be used to measure O3 levels. In summary, the proposed studies will enable us to develop a safe and reliable method of NO production that increases the accessibility and therapeutic uses of this life saving therapy.